肝细胞来源外泌体通过LncRNA H19促进酒精性肝纤维化血管再生的作用研究

Angiogenesis induced by liver sinusoidal endothelial cells (LSEC) is an important mechanism of alcoholic hepatic fibrosis (AHF). Exosomes, the important media in cell-to-cell communication, are now recognized to play a critical role in liver physiology and pathophysiology. In early studies, we found that hepatocytes released exosomes in response to alcohol can stimulate angiogenesis. The Arraystar human LncRNA V4.0 microarray showed that LncRNA H19 was up-regulated in hepatocyte derived exosomes. However, the expression of LncRNA H19 was significantly increased and correlated with abnormal liver angiogenesis positively in AHF’ liver tissue. LncRNAs were described to mediate tumour related angiogenesis by functioning as a competing endogenous RNA (ceRNA). The hypothesis of this project is that the exosomes released from hepatocytes contains LncRNA H19, they can stimulate angiogenesis by acting as ceRNA in AHF. This project aims to employ various cells, Cre-lox AHF mouse models and clinical specimens, using RNA immunoprecipitation, dual-luciferase reporter assay, laser capture microdissection and other technologies, to clarify the mechanism that alcohol stimulates hepatocytes to release LncRNA H19 containing exosomes. The exsomes transmit LncRNA H19 into LSEC. After that, LncRNA H19 stimulates LSEC angiogenesis by acting as ceRNA. This study will contribute to the understanding of angiogenesis mechanism in AHF, and provide new evidences for diagnosis and therapeutic targets of AHF.

肝窦内皮细（LSEC）异常血管再生在促进酒精性肝纤维化（AHF）发展中起重要作用。外泌体作为细胞间信息传递的方式，参与肝纤维化的病理生理过程。申请者前期发现，酒精诱导肝细胞释放外泌体可促进血管新生；LncRNA芯片显示外泌体中H19明显升高；LncRNA H19在AHF肝组织中升高并与血管增生呈正相关。文献报道，LncRNA可作为竞争性内源RNA（ceRNA）参与肿瘤血管生成。申请者提出，肝细胞来源外泌体携带的LncRNA H19可通过ceRNA机制促进LSEC介导的AHF血管再生。本研究拟通过多种细胞、Cre-lox酒精性肝纤维化小鼠等模型和临床标本，采用RNA免疫沉淀、双萤光素酶报告基因检测、激光捕获显微切割等技术，探讨肝细胞来源外泌体将LncRNA H19传递至LSEC内，通过ceRNA模式促进血管再生的分子机制。该研究将为AHF血管再生机制提供新观点，为诊断及治疗AHF提供新靶点。

酒精性肝纤维化（AHF）的发病机制主要包括纤维结缔组织的异常沉积和血管异常再生。我们前期研究发现，酒精可诱导肝细胞释放外泌体增加，导致肝脏炎症的发生，还可促进内皮细胞血管再生。通过LncRNA芯片分析，我们发现促进内皮细胞血管再生的外泌体中LncRNA H19明显升高。此外，在AHF患者肝脏组织中，LncRNA H19升高并与血管再生呈正相关。以上提示，外泌体相关LncRNA H19在调控AHF血管再生过程中发挥重要作用。该研究旨在进一步探讨酒精刺激肝细胞释放的外泌体中LncRNA H19调控肝纤维化血管异常再生的具体作用机制。收集酒精性肝纤维化及正常肝组织进行免疫组化学染色并计算微血管密度；采用激光捕获显微切割技术提取正常及AHF患者LSEC，检测LncRNA H19、VEGFA的表达情况，结果证明LncRNA H19与肝纤维化血管再生程度成正相关。采用“白酒-吡唑-植物油”混合液灌胃法复制动物模型,通过HE和Massion染色评价模型组与对照组肝脏纤维化程度，Western blot、Real-time PCR检测并分析LSEC中LncRNA H19、VEGFA的表达及相关性，得到相同结论。体外通过酒精刺激肝细胞提取的外泌体与非酒精来源外泌体染色后与内皮细胞共孵育，发现外泌体刺激使血管成管能力明显增强。通过生物信息学数据库分析，我们发现LncRNA H19可能结合的相关miRNA有39个，其中能与VEGFA的3'UTRs结合并具有相同MREs的miRNA有10个，进一步筛选出8个具有调控血管再生作用的相关miRNA。最后通过过表达不同肝细胞中LncRNA H19，酒精刺激后提取外泌体，与关键miRNA过表达的LSEC共培养，验证血管再生程度。总之，LncRNA H19作为ceRNA，通过关键miRNA调控相关靶基因的表达，促进血管再生。